Mouse hepatitis virus (MHV) uses its N-terminal domain (NTD) of the viral spike (S) protein to bind the host receptor mouse carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a) and mediate virus entry. Our previous crystal structure study of the MHV NTD/mCEACAM1a complex (G. Peng, D. Sun, K. R. Rajashankar, Z. Qian, et al., Proc Natl Acad Sci U S A 108:10696-10701, 2011, https://doi.org/10.1073/pnas.1104306108) reveals that there are 14 residues in the NTD interacting with the receptor. However, their contribution to receptor binding and virus entry has not been fully investigated. Here we analyzed 13 out of 14 contact residues by mutagenesis and identified I22 as being essential for receptor binding and virus entry. Unexpectedly, we found that G29 was critical for the conformational changes of the S protein triggered by either receptor binding or high pH. Replacement of G29 with A, D, F, K, M, and T, to different extents, caused spontaneous dissociation of S1 from the S protein, resulting in an enhancement of high-pH-triggered receptor-independent syncytium (RIS) formation in HEK293T cells, compared to the wild type (WT). In contrast, replacement of G29 with P, a turn-prone residue with a strict conformation, hindered virus entry and conformational changes of the S protein triggered by either receptor binding or pH 8.0, suggesting that the structural turn around G29 and its flexibility are critical. Finally, stabilization of the NTD by G29P had almost no effect on pH-independent RIS induced by the Y320A mutation in the C-terminal domain (CTD) of the S1 subunit, indicating that there might be an absence of cross talk between the NTD and CTD during conformational changes of the S protein. Our study will aid in better understanding the mechanism of how conformational changes of the S protein are triggered.IMPORTANCE Binding of the MHV S protein to the receptor mCEACAM1a triggers conformational changes of S proteins, leading to the formation of a six-helix bundle and viral and cellular membrane fusion. However, the mechanism by which the conformational change of the S protein is initiated after receptor binding has not been determined. In this study, we showed that while replacement of G29, a residue at the edge of the receptor binding interface and the center of the structural turn after the β1-sheet of the S protein, with D or T triggered spontaneous conformational changes of the S protein and pH-independent RIS, the G29P mutation significantly impeded the conformational changes of S proteins triggered by either receptor binding or pH 8.0. We reason that this structural turn might be critical for conformational changes of the S protein and that altering this structural turn could initiate conformational changes of the S protein, leading to membrane fusion.

ABSTRACT The spike glycoprotein (S) of murine coronavirus mouse hepatitis virus (MHV) strain A59 uses murine carcinoembryonic antigen-related cell adhesion molecule 1a as its receptor for cell entry, but S protein can also be triggered in the absence of receptor by pH 8.0 alone at 37°C. The mechanism by which conformational changes of this S glycoprotein can be triggered by pH 8.0 has not yet been determined. Here, we show that MHV-A59 S protein is triggered by pH 8.0 at 37°C to induce receptor-independent syncytium (RIS) formation on 293T cells, and that the conformational changes in S proteins triggered by pH 8.0 are very similar to those triggered by receptor binding. We systemically mutated each of 15 histidine residues in S protein and found that H209 is essential for pH 8.0-triggered RIS formation, while H179, H441, H643, and H759 also play important roles in this process. Replacement of H209 with Ala had no effect on receptor binding, but in murine 17Cl.1 cells mutant H209A MHV-A59 showed delayed growth kinetics and was readily outcompeted by wild-type virus when mixed together, indicating that the H209A mutation caused a defect in virus fitness. Finally, the H209A mutation significantly increased the thermostability of S protein in its prefusion conformation, which may raise the energy barrier for conformational change of S protein required for membrane fusion and lead to a decrease in virus fitness in cell culture. Thus, MHV-A59 may have evolved to lower the stability of its S protein in order to increase virus fitness. IMPORTANCE Enveloped viruses enter cells through fusion of viral and cellular membranes, and the process is mediated by interactions between viral envelope proteins and their host receptors. In the prefusion conformation, viral envelope proteins are metastable, and activation to the fusion conformation is tightly regulated, since premature activation would lead to loss of viral infectivity. The stability of viral envelope proteins greatly influences their activation and virus fitness. Here, we report that, similar to the A82V mutation in Ebola glycoprotein, in the S glycoprotein of murine coronavirus MHV-A59, the histidine residue at position of 209 significantly affects the thermal stability of the S protein, determines whether S protein can be activated at 37°C by either pH 8.0 alone or by receptor binding, and affects viral fitness in cell culture. Thus, the spike glycoprotein of MHV-A59 has evolved to retain histidine at position 209 to optimize virus fitness.

Identification of H209 essential for pH 8-triggered receptor-independent syncytium 1 formation by S protein of MHV-A59 2 Pei Li, Yiwei Shan, Wangliang Zheng, Xiuyuan Ou, Dan Mi, Zhixia Mu, 3 Kathryn V Holmes, Zhaohui Qian 4 MOH Key laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, 5 Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 6 100176, China; Department of Microbiology, University of Colorado School of 7 Medicine, Aurora, CO 80045, USA 8

This study analyzed the impact of sex, hemodynamic profile and valve fusion pattern on aortopathy associated with bicuspid aortic valve (BAV). The NHLBI GenTAC Registry provided comprehensive information on a large population of well-characterized patients with BAV. Of 969 enrolled patients with BAV, 551 (57%; 77% male) had already undergone valvular and/or aortic surgery. Echocardiographic imaging data were available on 339 unoperated or pre-operative participants who form the basis of this study. BAV function was normal in 45 (14%), with predominant regurgitation (AR) in 127 (41%), and predominant stenosis (AS) in 76 (22%). Moderate-severe AR was associated with larger sinuses of Valsalva (SOV) diameters compared to normal function and AS (all p<0.01). Moderate-severe AS was associated with larger AscAo diameter compared to normal function (p=0.003) but not to AR. SOV diameter was larger in men than women (3.7±0.7 vs. 3.3±0.6 cm, p<0.0001), whereas ascending aortic (AscAo) diameters were comparable (3.9±0.9 vs. 3.7±0.9 cm, p=0.08). Right-left commissural fusion was associated with larger SOV diameter (3.7±0.7 vs. 3.3±0.6 cm, p<0.0001) compared to right-noncoronary fusion pattern. Predominant AR was more common in men (45% vs. 27%, p=0.004) whereas AS was more common in women (29% vs. 18%, p=0.04). In conclusion, in the GenTAC Registry, AR was associated with diffuse (annular, SOV and AscAo) enlargement whereas moderate-severe AS was only associated with AscAo enlargement. Male sex and right-left cusp pattern of cusp fusion were associated with larger SOV diameters and greater likelihood of AR, whereas females had a higher prevalence of AS.

We have utilized a de novo designed two-stranded α-helical coiled-coil template to display conserved α-helical epitopes from the stem region of hemagglutinin (HA) glycoproteins of influenza A. The immunogens have all the surface-exposed residues of the native α-helix in the native HA protein of interest displayed on the surface of the two-stranded α-helical coiled-coil template. This template when used as an immunogen elicits polyclonal antibodies which bind to the α-helix in the native protein. We investigated the highly conserved sequence region 421-476 of HA by inserting 21 or 28 residue sequences from this region into our template. The cross-reactivity of the resulting rabbit polyclonal antibodies prepared to these immunogens was determined using a series of HA proteins from H1N1, H2N2, H3N2, H5N1, H7N7, and H7N9 virus strains which are representative of Group 1 and Group 2 virus subtypes of influenza A. Antibodies from region 449-476 were Group 1 specific. Antibodies to region 421-448 showed the greatest degree of cross-reactivity to Group 1 and Group 2 and suggested that this region has a great potential as a "universal" synthetic peptide vaccine for influenza A. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 144-159, 2016.

Hosts and pathogens are locked in an evolutionary arms race. To infect mice, mouse hepatitis coronavirus (MHV) has evolved to recognize mouse CEACAM1a (mCEACAM1a) as its receptor. To elude MHV infections, mice may have evolved a variant allele from the Ceacam1a gene, called Ceacam1b, producing mCEACAM1b, which is a much poorer MHV receptor than mCEACAM1a. Previous studies showed that sequence differences between mCEACAM1a and mCEACAM1b in a critical MHV-binding CC' loop partially account for the low receptor activity of mCEACAM1b, but detailed structural and molecular mechanisms for the differential MHV receptor activities of mCEACAM1a and mCEACAM1b remained elusive. Here we have determined the crystal structure of mCEACAM1b and identified the structural differences and additional residue differences between mCEACAM1a and mCEACAM1b that affect MHV binding and entry. These differences include conformational alterations of the CC' loop as well as residue variations in other MHV-binding regions, including β-strands C' and C'' and loop C'C''. Using pseudovirus entry and protein-protein binding assays, we show that substituting the structural and residue features from mCEACAM1b into mCEACAM1a reduced the viral receptor activity of mCEACAM1a, whereas substituting the reverse changes from mCEACAM1a into mCEACAM1b increased the viral receptor activity of mCEACAM1b. These results elucidate the detailed molecular mechanism for how mice may have kept pace in the evolutionary arms race with MHV by undergoing structural and residue changes in the MHV receptor, providing insight into this possible example of pathogen-driven evolution of a host receptor protein.

BACKGROUND Aortic dissection (AoD) is a serious complication of thoracic aortic aneurysm (TAA). Relative risk for AoD in relation to TAA etiology, incidence, and pattern after prophylactic TAA surgery are poorly understood.OBJECTIVES This study sought to determine the incidence, pattern, and relative risk for AoD among patients with genetically associated TAA.METHODS The population included adult GenTAC participants without AoD at baseline. Standardized core laboratory tests classified TAA etiology and measured aortic size. Follow-up was performed for AoD.RESULTS Bicuspid aortic valve (BAV) (39%) and Marfan syndrome (MFS) (22%) were the leading diagnoses in the studied GenTAC participants (n = 1,991). AoD occurred in 1.6% over 3.6 +/- 2.0 years; 61% of AoD occurred in patients with MFS. Cumulative AoD incidence was 6-fold higher among patients with MFS (4.5%) versus others (0.7%; p < 0.001). MFS event rates were similarly elevated versus those in patients with BAV (0.3%; p < 0.001). AoD originated in the distal arch or descending aorta in 71%; 52% of affected patients, including 68% with MFS, had previously undergone aortic grafting. In patients with proximal aortic surgery, distal aortic size (descending thoracic, abdominal aorta) was larger among patients with AoD versus those without AoD (both p < 0.05), whereas the ascending aorta size was similar. Conversely, in patients without previous surgery, aortic root size was greater in patients with subsequent AoD (p < 0.05), whereas distal aortic segments were of similar size. MFS (odds ratio: 7.42; 95% confidence interval: 3.43 to 16.82; p < 0.001) and maximal aortic size (1.86 per cm; 95% confidence interval: 1.26 to 2.67; p = 0.001) were independently associated with AoD. Only 4 of 31 (13%) patients with AoD had pre-dissection images that fulfilled size criteria for prophylactic TAA surgery at a subsequent AoD site.CONCLUSIONS Among patients with genetically associated TAA, MFS augments risk for AoD even after TAA grafting. Although increased aortic size is a risk factor for subsequent AoD, events typically occur below established thresholds for prophylactic TAA repair. (C) 2016 by the American College of Cardiology Foundation.

ABSTRACT The fusion peptides (FP) play an essential role in fusion of viral envelope with cellular membranes. The location and properties of the FPs in the spike (S) glycoproteins of different coronaviruses (CoV) have not yet been determined. Through amino acid sequence analysis of S proteins of representative CoVs, we identified a common region as a possible FP (pFP) that shares the characteristics of FPs of class I viral fusion proteins, including high Ala/Gly content, intermediate hydrophobicity, and few charged residues. To test the hypothesis that this region contains the CoV FP, we systemically mutated every residue in the pFP of Middle East respiratory syndrome betacoronavirus (MERS-CoV) and found that 11 of the 22 residues in the pFP (from G953 to L964, except for A956) were essential for S protein-mediated cell-cell fusion and virus entry. The synthetic MERS-CoV pFP core peptide ( 955 IAGVGWTAGL 964 ) induced extensive fusion of liposome membranes, while mutant peptide failed to induce any lipid mixing. We also selectively mutated residues in pFPs of two other β-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and mouse hepatitis virus (MHV). Although the amino acid sequences of these two pFPs differed significantly from that of MERS-CoV and each other, most of the pFP mutants of SARS-CoV and MHV also failed to mediate membrane fusion, suggesting that these pFPs are also the functional FPs. Thus, the FPs of 3 different lineages of β-CoVs are conserved in location within the S glycoproteins and in their functions, although their amino acid sequences have diverged significantly during CoV evolution. IMPORTANCE Within the class I viral fusion proteins of many enveloped viruses, the FP is the critical mediator of fusion of the viral envelope with host cell membranes leading to virus infection. FPs from within a virus family, like influenza viruses or human immunodeficiency viruses (HIV), tend to share high amino acid sequence identity. In this study, we determined the location and amino acid sequences of the FPs of S glycoproteins of 3 β-CoVs, MERS-CoV, SARS-CoV, and MHV, and demonstrated that they were essential for mediating cell-cell fusion and virus entry. Interestingly, in marked contrast to the FPs of influenza and HIV, the primary amino acid sequences of the FPs of β-CoVs in 3 different lineages differed significantly. Thus, during evolution the FPs of β-CoVs have diverged significantly in their primary sequences while maintaining the same essential biological functions. Our findings identify a potential new target for development of drugs against CoVs.

BackgroundThe risk of aortic complications associated with pregnancy in women with Marfan syndrome (MFS) is not fully understood. Methods and ResultsMFS women participating in the large National Registry of Genetically Triggered Thoracic Aortic Aneurysms and Cardiovascular Conditions (GenTAC) were evaluated. Among 184 women with MFS in whom pregnancy information was available, 94 (51%) had a total of 227 pregnancies. Among the women with pregnancies, 10 (10.6%) experienced a pregnancy‐related aortic complication (4 type A and 3 type B dissections, 1 coronary artery dissection, and 2 with significant [≥3 mm] aortic growth). Five of 7 aortic dissections, including all 3 type B, and the coronary dissection (75% of all dissections) occurred in the postpartum period. Only 5 of 8 women with pregnancy‐associated dissection were aware of their MFS diagnosis. The rate of aortic dissection was higher during the pregnancy and postpartum period (5.4 per 100 person‐years vs 0.6 per 100 person‐years of nonpregnancy; rate ratio, 8.4 [95% CI=3.9, 18.4]; P<0.0001). ConclusionsPregnancy in MFS is associated with an increased risk of aortic dissection, both types A and B, particularly in the immediate postpartum period. Lack of knowledge of underlying MFS diagnosis before aortic dissection is a major contributing factor. These findings underscore the need for early diagnosis, prepregnancy risk counseling, and multidisciplinary peripartum management.

Background: The influence of gender and age on phenotypic features of individuals with Marfan syndrome has not been systematically examined in a large cohort of both children and adults. Methods: We evaluated 789 Marfan patients enrolled in the NHLBI GenTAC Registry (53% male; mean age 31 [range: 1-86 years]). Females aged≥15 and males aged≥16 years were considered adults based on average age of skeletal maturity. Results: Children (n=183) and adults (n=606) (see Table) were comparable in revised Ghent systemic score, ectopia lentis, and most phenotypic features, including prevalences of aortic root dilatation and mitral prolapse. However, adults were more likely to have spontaneous pneumothorax, scoliosis, dural ectasia and striae. Prophylactic aortic root replacement and mitral valve surgery were rare during childhood vs. adulthood (2 vs. 35% and 1 vs. 9%, respectively, both p<0.0001). There was only one childhood aortic dissection (type B in 15 year old male). Overall females were more likely than males to have arachnodactyly (54 vs. 40%), scoliosis (60 vs. 49%), and dural ectasia (16 vs. 10%), all p<0.01. Adult males were more likely to have aortic root dilatation (92 vs. 84%), aortic regurgitation (55 vs. 36%) and to have undergone prophylactic aortic root replacement (47 vs. 24%), all p<0.001. Prevalence of prior aortic dissection was higher in males than females (25 vs. 18%, p=0.06), 44% of which were type B. Mean age at dissection (35 vs. 38 years), age at prophylactic surgery (32 vs. 35 years), and need for mitral valve surgery (9.5 vs. 10.0%) were similar between men and women, respectively. Conclusions: Pulmonary, skeletal and aortic complications, but not other phenotypic features, increase with aging in Marfan syndrome. Aortic aneurysms and prophylactic aortic surgery are more common in men. In the era of current surgical and medical treatment, aortic dissection, commonly type B, occurs in an appreciable proportion of Marfan patients, especially among men.

Background: Definition of pregnancy risk in women with Marfan syndrome (MFS) has been confounded by ascertainment bias, lack of imaging data, small sample size, and inclusion of women without a known MFS diagnosis before a pregnancy complication. To lessen these limitations, we analyzed MFS women participating in the large GenTAC Registry. Results: Among 184 women in whom pregnancy information was available, 94 women (51%) had a total of 227 pregnancies (average 2.5, range 1-6). Compared to MFS women who never became pregnant, those with pregnancies were older at time of MFS diagnosis (27 vs. 13 years, p<0.0001) and at time of GenTAC enrollment (47 vs. 37 years, p<0.0001). When comparing ever-pregnant to never-pregnant women with MFS at the time of registry entry, higher historical rates of aortic dissection and prophylactic proximal aortic surgery were observed (24/94 [25.5%] vs. 13/90 [14.4%], p=0.068 and 15/94 [16.0%] vs. 4/90 [4.4%], p=0.014; respectively). Among the 94 MFS women with pregnancies, 10 (10.6%) experienced a pregnancy-related aortic complication (4 Type A and 3 Type B dissections, one coronary artery dissection, and 2 with significant [≥3 mm] aortic growth, 1 of whom required post-partum prophylactic surgery). Four of the aortic dissections, including all 3 Type B, and the coronary dissection occurred in the post-partum period. Only 3 of 7 women (43%) with an aortic dissection related to pregnancy were aware of their MFS diagnosis before pregnancy. Women with and without pregnancy-associated aortic complications did not differ in phenotypic features. The rate of aortic dissection was higher during pregnancy + post-partum period (3.5 per 100 person-years vs. 0.8 per 100 person-years, odds ratio 4.6 [95% confidence interval 2.1-10.1], p=0.0002). Conclusions: Pregnancy in MFS is associated with an increased risk of aortic dissection, both Types A and B, particularly in the immediate post-partum period, and more frequent prophylactic surgery. Lack of knowledge of underlying MFS diagnosis before aortic dissection is a major contributing factor. These findings underscore the need for early diagnosis, pre-pregnancy risk counselling, and multi-disciplinary peri-partum management.

Background: Aortic aneurysm phenotype in bicuspid aortic valve (BAV) may vary according to gender, hemodynamic alterations, and valve morphology (type of cusp fusion). To better understand these potential associations, BAV participants in the GenTAC Registry were analyzed. Methods: 973 patients with BAV were enrolled (76.3% male; mean age 44.5 years). At time of enrollment, 549 (56.4%; 76.9% male) had undergone valvular and/or aortic surgery. Imaging data were available on 424 unoperated or pre-operative participants. Results: Sinuses of Valsalva (SOV) diameter was larger in men than women (3.90 vs. 3.36 cm, p<0.0001); Z score: 2.2±2.3 vs. 1.4±1.9, p=0.002), whereas ascending aortic (AscAo) diameters were comparable (4.01 vs. 3.89, p=0.22; Z score: 3.2±2.0 vs. 3.5±2.2, p=0.124). BAV function was normal in 15.0%, predominantly regurgitant (AR) in 38.9%, predominantly stenotic (AS) in 21.6%, and mixed AS/AR in 16.0%. AR was more common in men (43.8 vs. 25.0%, p=0.003) whereas AS was more common in women (30.0 vs. 18.6%, p=0.033). Moderate-severe AR was associated with larger SOV diameters than mild AS (p=0.01) or moderate-severe AS (p=0.0006) or normal function (p<0.001). SOV diameters in mild AR were larger than those with normal function (p=0.012). Moderate-severe AS was associated with significantly larger AscAo diameter compared to normal function (p=0.002) but not compared to BAV with AR. AscAo diameters were larger in AR compared to normal function (both p<0.001). Diameters of the SOV (3.70±0.67 vs. 3.36±0.56 cm, p=0.001) were larger in right-left fusion than in right-non fusion pattern whereas AscAo diameter was comparable. Conclusions: In the GenTAC Registry, SOV diameters were larger in males, even after adjustment for body size. AR was associated with both SOV and AscAo enlargement whereas moderate-severe AS was only associated with AscAo enlargement. Aortic diameters were larger in those with right-left cusp fusion independent of valve function.

ABSTRACT Coronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (β-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas the β-CoVs severe acute respiratory syndrome CoV in group B and Middle East respiratory syndrome CoV in group C and several α-CoVs use the downstream C domain in their S proteins to recognize their receptor proteins. To identify the receptor-binding domain in the spike of human β-CoV HKU1 in group A, we generated and mapped a panel of monoclonal antibodies (MAbs) to the ectodomain of HKU1 spike protein. They did not cross-react with S proteins of any other CoV tested. Most of the HKU1 spike MAbs recognized epitopes in the C domain between amino acids 535 and 673, indicating that this region is immunodominant. Two of the MAbs blocked HKU1 virus infection of primary human tracheal-bronchial epithelial (HTBE) cells. Preincubation of HTBE cells with a truncated HKU1 S protein that includes the C domain blocked infection with HKU1 virus, but preincubation of cells with truncated S protein containing only the NTD did not block infection. These data suggest that the receptor-binding domain (RBD) of HKU1 spike protein is located in the C domain, where the spike proteins of α-CoVs and β-CoVs in groups B and C bind to their specific receptor proteins. Thus, two β-CoVs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins. IMPORTANCE Mouse hepatitis virus, a β-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another β-CoV in group A, uses the NTD to bind to its sialic-acid containing receptor. In marked contrast, the NTD of the spike glycoprotein of human respiratory β-CoV HKU1, which is also in group A, does not bind sugar. In this study, we showed that for the spike protein of HKU1, the purified C domain, downstream of the NTD, could block HKU1 virus infection of human respiratory epithelial cells, and that several monoclonal antibodies that mapped to the C domain neutralized virus infectivity. Thus, the receptor-binding domain of HKU1 spike glycoprotein is located in the C domain. Surprisingly, two β-CoVs in group A, mouse hepatitis virus and HKU1, have evolved to use different regions of their spike glycoproteins to recognize their respective receptors.

The recent discovery of genetically distinct hantaviruses in multiple species of shrews and moles prompted a further exploration of their host diversification by analyzing frozen, ethanol-fixed and RNAlater®-preserved archival tissues and fecal samples from 533 bats (representing seven families, 28 genera and 53 species in the order Chiroptera), captured in Asia, Africa and the Americas in 1981–2012, using RT-PCR. Hantavirus RNA was detected in Pomona roundleaf bats (Hipposideros pomona) (family Hipposideridae), captured in Vietnam in 1997 and 1999, and in banana pipistrelles (Neoromicia nanus) (family Vespertilionidae), captured in Côte d’Ivoire in 2011. Phylogenetic analysis, based on the full-length S- and partial M- and L-segment sequences using maximum likelihood and Bayesian methods, demonstrated that the newfound hantaviruses formed highly divergent lineages, comprising other recently recognized bat-borne hantaviruses in Sierra Leone and China. The detection of bat-associated hantaviruses opens a new era in hantavirology and provides insights into their evolutionary origins.

From 1 January 2009 to 31 May 2013, 15 287 respiratory specimens submitted to the Clinical Virology Laboratory at the Children's Hospital Colorado were tested for human coronavirus RNA by reverse transcription-PCR. Human coronaviruses HKU1, OC43, 229E and NL63 co-circulated during each of the respiratory seasons but with significant year-to-year variability, and cumulatively accounted for 7.4-15.6 % of all samples tested during the months of peak activity. A total of 79 (0.5 % prevalence) specimens were positive for human betacoronavirus HKU1 RNA. Genotypes HKU1 A and B were both isolated from clinical specimens and propagated on primary human tracheal-bronchial epithelial cells cultured at the air-liquid interface and were neutralized in vitro by human intravenous immunoglobulin and by polyclonal rabbit antibodies to the spike glycoprotein of HKU1. Phylogenetic analysis of the deduced amino acid sequences of seven full-length genomes of Colorado HKU1 viruses and the spike glycoproteins from four additional HKU1 viruses from Colorado and three from Brazil demonstrated remarkable conservation of these sequences with genotypes circulating in Hong Kong and France. Within genotype A, all but one of the Colorado HKU1 sequences formed a unique subclade defined by three amino acid substitutions (W197F, F613Y and S752F) in the spike glycoprotein and exhibited a unique signature in the acidic tandem repeat in the N-terminal region of the nsp3 subdomain. Elucidating the function of and mechanisms responsible for the formation of these varying tandem repeats will increase our understanding of the replication process and pathogenicity of HKU1 and potentially of other coronaviruses.